Introduction: Properties and functionality of human tissues and organs arise from their well-controlled three-dimensional (3D) organisation. The ability to engineer 3D environments with tuneable, hierarchical, and anisotropic molecular composition would be highly beneficial in areas such as tissue engineering, regenerative medicine, developmental biology, or drug development. Here we present a simple and affordable hydrogel fabrication method to print different functional molecules (i.e. peptides, proteins) with high precision and tuneability within 3D hydrogels in order to engineer complex hydrogel environments capable of guiding cell growth and recreating biological scenarios.
Materials and Methods: The fabrication process relies on the capacity to move charged molecules using electric fields (electrophoresis) that are controlled by a porous membrane (mask) that defines the regions where the printing takes place. By simple modification of the electric field, the mask, and the loading parameters, it is possible to controllably tune the printing process and the complexity of the final hydrogel patterns. The technique is highly versatile as it permits patterning different types of hydrogels (i.e. agarose, collagen, polyacrylamide, poly(ethylene-glycol)diacrylate, and their blends) and functional molecules (i.e. elastin, immunoglobulins, and fibronectin).
Results and Discussion: We have successfully generated patterns of proteins of different molecular weights (~63-kDa elastin - 450kDa fibronectin) within different readily available hydrogels. High precision patterns were printed in the form of linear, bending, widening and converging tracks, as well as gradients of multiple molecules. The patterns were generated on large surface areas (up to ~1cm radius) with great depth (up to ~2cm), resolution (30-150 micrometres in width) and aspect ratio (width-to-depth ratio). Preliminary cell culture studies (NIH 3T3 fibroblasts) revealed selective molecular recognition inducing cell penetration into polyacrylamide-gelatine hydrogel blend, demonstrating that the patterned molecules are functional after the fabrication process. The novel technique is simple, tuneable, can be used with any type of hydrogel, can print different molecules including whole proteins, and can generate molecular patterns from tens of microns up to centimetres in size and depth. These capabilities provide advantages over other techniques for the fabrication of anisotropic hydrogels to recreate control biological environments and processes with high spatial control.
Conclusion: The capacity to manipulate and localize multiple proteins in their native state replicating geometrical configurations found in biological systems would benefit applications that require fine control of molecular and cell organisation. We believe that our technique has the potential to grow into a novel hydrogel fabrication platform for the generation of multifunctional environments with improved complexity for biomimetic cell culture studies.
European Research Council (ERC); Queen Mary Innovation Ltd